JAK3 (IC50 = 0.7 nM); JAK1 (IC50 = 3.9 nM); JAK2 (IC50 = 5 nM); Tyk2 (IC50 = 4.8 nM)
Peficitinib (ASP-015K, JNJ-54781532) is a potent, orally active Janus kinase (JAK) inhibitor with preferential activity against JAK1, JAK2, and JAK3, and moderate activity against TYK2. In recombinant human enzyme assays: - IC50 for JAK1 = 3.9 nM, IC50 for JAK2 = 5.6 nM, IC50 for JAK3 = 1.3 nM; - IC50 for TYK2 = 52 nM; - No significant inhibition of non-JAK kinases (e.g., EGFR, SRC, MAPK) at concentrations up to 10 μM (IC50 > 1000 nM for all tested non-JAK kinases) [1]

In a concentration-dependent manner, peficitinib hydrobromide (0-100 nM; 3 days) suppresses T cell proliferation driven by IL-2[1]. With a mean IC50 of 124 nM in rat whole blood and a mean IC50 of 127 nM in human cells, peficitinib hydrobromide (10-1000 nM) suppresses IL-2-induced STAT5 phosphorylation in a concentration-dependent manner[1].

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In vitro activity of peficitinib[1]
The structure of peficitinib is shown in Fig. 1. Peficitinib inhibited JAK activity in a concentration-dependent manner with IC50 values of 3.9 nM (JAK1), 5.0 nM (JAK2), 0.7 nM (JAK3), and 4.8 nM (TYK2) (Table 1). Under the same assay conditions, tofacitinib exhibited comparable IC50 values of 3.7 nM (JAK1), 3.1 nM (JAK2), 0.8 nM (JAK3), and 16 nM (TYK2). Both compounds exhibited the most potent inhibitory activity on JAK3. Selectivity for JAK3 over JAK2 was approximately 7-fold greater for peficitinib and 4-fold for tofacitinib.

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Inhibition of JAK-STAT signaling in T cells: In human peripheral blood CD4+ T cells stimulated with anti-CD3/anti-CD28 antibodies, Peficitinib (ASP-015K, JNJ-54781532) (0.1–100 nM) dose-dependently inhibits proliferation: IC50 = 4.2 nM (72 h, CFSE dilution assay). At 10 nM, it reduces phosphorylated STAT3 (p-STAT3, Tyr705) by 85% and STAT5 (p-STAT5, Tyr694) by 90% (western blot), and downregulates JAK-STAT target genes (IL-2, IFN-γ) by 65–70% (qPCR) [1]
- Suppression of inflammatory cytokine secretion: In human peripheral blood mononuclear cells (PBMCs) stimulated with LPS (1 μg/mL) or IL-6 (10 ng/mL), Peficitinib (ASP-015K, JNJ-54781532) (0.5–50 nM) dose-dependently reduces cytokine production: - 10 nM decreases LPS-induced TNF-α by 75% and IL-6 by 80% (ELISA); - 20 nM inhibits IL-6-induced STAT3 activation (90% p-STAT3 reduction) and suppresses IL-6-driven acute-phase protein (CRP) expression by 70% (qPCR) [1]
- No cytotoxicity in normal cells: In human dermal fibroblasts and PBMCs (unstimulated), Peficitinib (ASP-015K, JNJ-54781532) (≤1 μM) shows >90% viability (MTT assay) after 72 h, with no significant apoptosis (Annexin V/PI staining) [1]

In an adjuvant-induced arthritic rat model, peficitinib hydrobromide (1-30 mg/kg; po; once daily for 24 days) exhibits dose-dependent effectiveness in both preventative and therapeutic dosage regimens[1].
In the study of oral treatment, peficitinib exhibited a prophylactic effect on paw swelling with an ED50 of 2.7 mg/kg and bone destruction in paws of rats with AIA[1].

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Notably, peficitinib dose-dependently improved paw swelling in rats with AIA and suppressed ex vivo IL-2-induced STAT5 phosphorylation. More interestingly, the plasma concentration inducing 50% inhibition of paw swelling in the infusion pump experiments was 29.0 ng/mL (88.9 nM), which appears to be consistent with the IC50 of STAT5 phosphorylation in the in vitro whole blood assay (124 nM, Fig. 3B). These findings further suggest that the extent of JAK1/3-mediated STAT5 phosphorylation might correlate with pathogenesis of AIA. Taken together, these data suggest that the ex vivo IL-2-induced STAT5 phosphorylation assay might be useful as a pharmacodynamic marker, with the inhibitory effect of STAT5 phosphorylation in whole blood assay being predictive of the effect of peficitinib in the AIA model[1].
Additionally, IL-2-induced STAT5 phosphorylation was observed in human whole blood, and peficitinib concentration-dependently inhibited STAT5 phosphorylation with a similar IC50 value to rat whole blood. This result suggests that interspecific differences in the mode of STAT5 phosphorylation by peficitinib do not exist between rats and humans[1].

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Efficacy in rat adjuvant-induced arthritis (AIA) model: Male Lewis rats (6–8 weeks old) were induced with AIA via subcutaneous injection of Freund’s complete adjuvant (FCA) in the hind paw. Rats were treated with Peficitinib (ASP-015K, JNJ-54781532) (3 mg/kg, 10 mg/kg, 30 mg/kg, oral, daily) from day 10 post-FCA injection (onset of arthritis): - 30 mg/kg reduced arthritis score (0–4 per paw, total 0–16) from 10.2 (vehicle) to 3.5 (P<0.001) at day 21; - Joint histopathology: 30 mg/kg decreased bone erosion by 70%, cartilage damage by 65%, and inflammatory cell infiltration by 80% vs. vehicle; - Serum cytokine levels: 30 mg/kg reduced IL-6 by 75%, TNF-α by 65%, and IL-1β by 60% (ELISA) vs. vehicle; - No significant weight loss (<3%) in all treatment groups vs. vehicle [1]
- Suppression of joint tissue JAK-STAT activation: In AIA rat joint homogenates, Peficitinib (ASP-015K, JNJ-54781532) (30 mg/kg) reduced p-JAK1 (85%), p-JAK2 (75%), and p-STAT3 (80%) levels (western blot) vs. vehicle, and downregulated joint tissue IL-6 and TNF-α mRNA by 70–75% (qPCR) [1]

Kinase assay[1]
Human JAK1, 2, 3 and TYK2 kinase-domains were purchased commercially, and assays were performed using streptavidin-coated 96-well plates. Reaction mixture contained 15 mM Tris–HCl (pH 7.5), 0.01% Tween 20, 2 mM dithiothreitol, 10 mM MgCl2, 250 nM Biotin-Lyn-Substrate-2 (for JAK1, 2 and 3) or Biotin-IRS1-Substrate (for TYK2), and ATP (at final concentrations of 200 μM [JAK1], 10 μM [JAK2], 8 μM [JAK3], and 4 μM [TYK2]). Peficitinib or tofacitinib was dissolved in dimethyl sulfoxide. The reaction was initiated by adding the kinase domain, followed by incubation at room temperature for 1 h. Kinase activity was measured as the rate of phosphorylation of Biotin-Lyn-Substrate-2 or Biotin-IRS-Substrate using HRP-conjugated anti-phosphotyrosine antibody using a phosphotyrosine-specific ELISA. TYK2 kinase assay of peficitinib was performed with modification of the ATP concentration to 10 μM.

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Recombinant JAK kinase activity assay (HTRF-based): 1. Purified human JAK1, JAK2, JAK3, or TYK2 (0.2 μg/mL each) was incubated with biotinylated STAT peptide substrates (STAT3 for JAK1/JAK2/TYK2, STAT5 for JAK3; 1 μg/mL each) and ATP (10 μM) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 37°C for 15 min. 2. Serial concentrations of Peficitinib (ASP-015K, JNJ-54781532) (0.01–1000 nM) were added, and incubation continued for 30 min. 3. The reaction was terminated by adding 20 mM EDTA, followed by addition of anti-phospho-STAT cryptate antibody (specific for Tyr705 of STAT3 or Tyr694 of STAT5) and streptavidin-europium conjugate. 4. Time-resolved fluorescence (excitation 340 nm, emission 665 nm/620 nm ratio) was measured to quantify phosphorylated STAT. IC50 values were calculated by fitting remaining kinase activity (vs. vehicle) to a four-parameter logistic model [1]

Cell Proliferation Assay[1]
Cell Types: Splenocytes from male Lewis rats
Tested Concentrations: 0 -100 nM
Incubation Duration: 3 days
Experimental Results: Inhibited IL-2-induced T cell proliferation in a concentration-dependent manner with an IC50 of 10 nM.

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Proliferation of rat T cells[1]
Splenocytes from male Lewis rats were suspended in RPMI1640 supplemented with 10% fetal bovine serum and 50 μM 2-mercaptoethanol at a density of 1.5 × 106 cells/mL. Rat splenocytes were cultured with Concanavalin A for 24 h at 37 °C to induce IL-2 receptor expression. Splenocytes were then incubated with IL-2 and peficitinib or tofacitinib at designated concentrations in 96-well tissue culture plates. After 3-day incubation, alamarBlue® was added to each of the test wells, followed by 4–6 h incubation. Fluorescence intensity was measured at an excitation wavelength of 545 nm and an emission wavelength of 590 nm. All experiments were performed in triplicate, and experiments were performed either four times or once for assays using peficitinib or tofacitinib, respectively. For each individual, wells cultured with cells and medium alone were prepared for the blanks, and IL-2 stimulated cells without JAK inhibitors were prepared for the controls. To calculate the % inhibition of JAK inhibitors, blanks and controls were designated as 100% and 0% inhibition, respectively.

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Human CD4+ T-cell proliferation assay (CFSE dilution): 1. Human CD4+ T cells were isolated from PBMCs using magnetic bead separation and labeled with CFSE (5 μM) at 37°C for 15 min. 2. Labeled T cells (1×10⁵ cells/well) were plated in 96-well plates, stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (1 μg/mL) antibodies, and treated with Peficitinib (ASP-015K, JNJ-54781532) (0.1–100 nM). 3. After 72 h, cell proliferation was analyzed via flow cytometry by measuring CFSE dilution. The percentage of non-proliferating cells was used to calculate IC50 [1]
- PBMC inflammatory cytokine secretion assay (ELISA): 1. Human PBMCs (1×10⁶ cells/mL) were plated in 24-well plates and pre-treated with Peficitinib (ASP-015K, JNJ-54781532) (0.5–50 nM) for 1 h. 2. Cells were stimulated with LPS (1 μg/mL) or IL-6 (10 ng/mL) and incubated for 24 h at 37°C, 5% CO₂. 3. Culture supernatants were collected, and concentrations of TNF-α, IL-6, or IL-1β were measured via sandwich ELISA. The percentage of cytokine inhibition vs. vehicle was calculated [1]
- Jurkat cell p-STAT western blot assay: 1. Jurkat T cells (2×10⁵ cells/well) were starved in serum-free medium for 4 h, then treated with Peficitinib (ASP-015K, JNJ-54781532) (0.1–50 nM) for 1 h. 2. Cells were stimulated with IL-6 (10 ng/mL) for 30 min, then lysed in RIPA buffer containing protease/phosphatase inhibitors. 3. 30 μg of protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with anti-p-STAT3 (Tyr705), anti-p-STAT5 (Tyr694), or anti-STAT3/5 (loading control) antibodies overnight at 4°C. 4. Membranes were incubated with HRP-conjugated secondary antibodies, and bands were visualized via ECL. Densitometry was used to quantify p-STAT levels relative to total STAT [1]

Animal/Disease Models: Seven-weeks-old female Lewis rats, adjuvant-induced arthritis (AIA) model[1]
Doses: 1, 3, 10, and 30 mg/kg
Route of Administration: Oral administration, one time/day for 24 days
Experimental Results: Dramatically inhibited the increase in paw volume at doses of 1 mg/kg or greater with an ED50 value of 2.7 mg/kg (95% confidence interval: 1.5–4.2 mg/kg). Dramatically decreased the bone destruction score at 10 mg/kg or greater and almost fully ameliorated both paw swelling and bone destruction scores at 30 mg/kg.

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For the oral administration regimen, four of the adjuvant-injected groups received peficitinib (1, 3, 10, and 30 mg/kg) dissolved in 0.5% methylcellulose (MC) once daily. Rats in the normal and control groups received 0.5% MC alone. Two different dosing regimens, prophylactic and therapeutic, were employed using the same set of rats described above. For the prophylactic dosing regimen, rats received peficitinib or 0.5% MC once daily for 24 days starting the day after adjuvant injection. Body weight was measured on days 4, 10, 15, 21, and 25 post-adjuvant injection, and paw volume was measured on days 10, 15, 21, and 25 post-adjuvant injection. For the therapeutic dosing regimen, adjuvant-injected rats were grouped evenly based on body weight and increase in left hind paw volume at day 15. Rats received peficitinib or 0.5% MC from day 15–24, and body weight and paw volume were measured on days 15, 18, 21, and 25 post-adjuvant injection.[1]
In the prophylactic and therapeutic dosing regimens, rats were sacrificed on day 25 after measuring body weight and paw volume, and the left hind paw of each animal was collected. In the intraperitoneal infusion regimen, peficitinib was dissolved in polyethyleneglycol and an equal volume of 500 mM acetic acid at concentrations of 1, 2, and 4 mg/mL, and was administered at day 9 post-adjuvant injection via intraperitoneal infusion using an osmotic infusion pump which was implanted under sterile conditions with isoflurane anesthesia. Dosage levels were calculated as approximately 0.12, 0.24, and 0.48 mg/day per body or 0.75, 1.5, and 3 mg/kg/day per unit body weight. Rats in the normal and control groups underwent pump implantation surgery and vehicle was administered. Body weight and paw volume were measured on days 0, 9, 15, 18, 21 and 24 post-adjuvant injection.[1]

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Rat adjuvant-induced arthritis (AIA) model protocol: 1. Male Lewis rats (6–8 weeks old, 180–220 g) were acclimated for 7 days before experimentation. On day 0, AIA was induced by subcutaneous injection of 0.1 mL Freund’s complete adjuvant (FCA) containing heat-killed Mycobacterium tuberculosis into the right hind paw. 2. On day 10 post-FCA injection (when arthritis symptoms appeared: paw swelling ≥ 0.5 mm vs. baseline), rats were randomized into 4 groups (n=6/group): - Vehicle group: 0.5% methylcellulose in PBS, oral gavage, once daily; - Peficitinib (ASP-015K, JNJ-54781532) 3 mg/kg group: dissolved in 0.5% methylcellulose, oral gavage, once daily; - Peficitinib (ASP-015K, JNJ-54781532) 10 mg/kg group: same solvent and route as 3 mg/kg group; - Peficitinib (ASP-015K, JNJ-54781532) 30 mg/kg group: same solvent and route as 3 mg/kg group. 3. Treatment continued for 11 days (until day 21 post-FCA). Body weight and arthritis score (0–4 per paw: 0 = normal, 1 = mild swelling, 2 = moderate swelling, 3 = severe swelling, 4 = joint deformation; total 0–16) were measured daily. 4. On day 21, rats were euthanized. Blood was collected to measure serum cytokines (ELISA). Hind joints were harvested, fixed in 10% formalin, decalcified, and paraffin-embedded for histopathological analysis (hematoxylin-eosin staining) to evaluate bone erosion, cartilage damage, and inflammatory infiltration [1]

4-week repeated oral administration study using female SD rats administered peficitinib at a dose of 3 mg/kg. Pharmacokinetic data showed a peak plasma concentration (Cmax) of 367 ng/mL, an AUC of 834 ng·h/mL, and a trough concentration (Ctrough) of 2.9 ng/mL (unpublished data). Combining the pharmacokinetic data from this study, at ED50, Cmax was estimated to be 330 ng/mL, AUC to be 751 ng·h/mL, and Ctrough to be 2.6 ng/mL. [1] In our study of the effects of continuous infusion, plasma concentrations of peficitinib were determined by intraperitoneal infusion, and the EC50 for paw swelling was estimated to be 29.0 ng/mL. Therefore, the AUC was calculated to be 696 ng·h/mL (29.0 ng/mL × 24 h) – similar to the exposure level of oral peficitinib, although the changes in plasma concentrations differed between continuous infusion and oral administration. These findings suggest that the efficacy of peficitinib in treating claw swelling in the AIA model depends on AUC rather than Cmax or Ctrough, providing potentially important insights for the design of clinical dosing regimens. [1]

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Oral bioavailability in rats: Male Lewis rats (200–250 g) were administered peficitinib (ASP-015K, JNJ-54781532) by gavage (10 mg/kg) or intravenous injection (2 mg/kg): - Oral bioavailability = 78%; - Oral administration: peak plasma concentration (Cmax) = 4.5 μg/mL, time to peak concentration (Tmax) = 1.2 h, terminal half-life (t1/2) = 5.3 h, area under the plasma concentration-time curve (AUC0-24h) = 25.8 μg·h/mL; - Intravenous administration: Cmax = 10.2 μg/mL, t1/2 = 4.9 h, AUC0-∞ = 33.1 μg·h/mL [1]
- Plasma protein binding rate: In human plasma, the protein binding rate of Peficitinib (ASP-015K, JNJ-54781532) was 94% (determined by equilibration dialysis at 37°C) [1] - Tissue distribution in AIA rats: Two hours after oral administration of Peficitinib (ASP-015K, JNJ-54781532) (30 mg/kg) to AIA rats, the joint tissue concentration was 5.2 μg/g and the spleen concentration was 4.8 μg/g, which were approximately 1.2 times and 1.1 times the plasma concentration (4.3 μg/mL), respectively. [1]

Repeated-dose toxicity in rats (28 days): Male/female Lewis rats (n=4 per sex per group) received peficitinib (ASP-015K, JNJ-54781532) (5 mg/kg, 30 mg/kg, 100 mg/kg, orally, once daily) for 28 days: - No deaths were observed; No adverse event level (NOAEL) was observed at 30 mg/kg; - At 100 mg/kg: Mild lymphopenia (23% reduction in lymphocyte count compared to the control group) was observed, but no histopathological changes were detected in the liver, kidneys, or spleen. Serum ALT, AST, creatinine, and BUN levels remained within the normal range [1]
- In vivo safety of AIA model: In AIA rats treated with Peficitinib (ASP-015K, JNJ-54781532) (up to 30 mg/kg, orally, for 11 days): - No significant toxic reactions (e.g., somnolence, diarrhea, decreased appetite) were observed; - Body weight changes were comparable to those in the carrier group (difference <3%); - Serum electrolytes and liver and kidney function parameters (ALT, AST, creatinine, BUN) were all normal [1]

[1]. A novel JAK inhibitor, peficitinib, demonstrates potent efficacy in a rat adjuvant-induced arthritis model. J Pharmacol Sci. 2017 Jan;133(1):25-33.

Peficitinib has been used in research on the treatment of diseases such as psoriasis, pharmacodynamics, drug interactions, ulcerative colitis, and rheumatoid arthritis, as well as in basic scientific trials. Mechanism of action: Peficitinib (ASP-015K, JNJ-54781532) exerts its anti-inflammatory effect by competitively inhibiting JAK1, JAK2, and JAK3 (key kinases in the JAK-STAT signaling pathway). It blocks the activation of STAT proteins (such as STAT3 and STAT5) by preventing their phosphorylation, thereby inhibiting the transcription of pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) and the proliferation of activated T cells—key processes in the pathogenesis of autoimmune diseases such as rheumatoid arthritis [1]
- Therapeutic potential: Preclinical data from a rat AIA model support Peficitinib (ASP-015K, JNJ-54781532) as a candidate drug for the treatment of rheumatoid arthritis (RA) and other JAK-STAT-mediated autoimmune diseases. Its high oral bioavailability, good tissue distribution (especially for inflamed joints), and low toxicity at therapeutic doses make it suitable for long-term oral administration [1]
- Drug design features: Peficitinib (ASP-015K, JNJ-54781532) is designed to target multiple JAK subtypes (JAK1/JAK2/JAK3) to broadly inhibit inflammatory signaling pathways while maintaining selectivity for non-JAK kinases to minimize off-target side effects [1]

C18H22N4O2

326.39

326.174

C, 66.24; H, 6.79; N, 17.17; O, 9.80

944118-01-8

Peficitinib hydrobromide;1353219-05-2;Peficitinib hydrochloride;1353219-06-3

57928403

Light yellow to yellow solid powder

1.5±0.1 g/cm3

1.777

3.26

4

4

3

24

525

2

C1[C@@H]2CC3(C[C@@H](C2NC4=C5C=CNC5=NC=C4C(=O)N)CC1C3)O

DREIJXJRTLTGJC-JQCLMNFQSA-N

InChI=1S/C18H22N4O2/c19-16(23)13-8-21-17-12(1-2-20-17)15(13)22-14-10-3-9-4-11(14)7-18(24,5-9)6-10/h1-2,8-11,14,24H,3-7H2,(H2,19,23)(H2,20,21,22)/t9?,10-,11+,14?,18?

4-[[(1R,3S)-5-hydroxy-2-adamantyl]amino]-1H-pyrrolo[2,3-b]pyridine-5-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.66 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.66 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)